Journal: bioRxiv

Article Title: Glucose selectively drives a rapid oxidative burst and immunometabolic reprogramming in human neutrophils during Mycobacterium tuberculosis infection

doi: 10.64898/2026.05.05.722986

Figure Lengend Snippet: | Intact Mtb triggers an immediate, glucose-dependent oxidative burst in human neutrophils . a , Workflow for isolating neutrophils from healthy donors and measuring neutrophil oxidative burst and other bioenergetic parameters during Mtb infection using the Agilent Seahorse XFe96 Extracellular Flux Analyzer. All XF data were normalized to 10,000 cells using Gen5 software on an Agilent BioTek Cytation 5 Multimode Reader. Serum-free RPMI 1640 medium was used in all assays. b , Representations of XF assay design, including injection strategies for measuring extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and oxidative burst. The magnitude of oxidative burst is determined by calculating the area under the OCR curve (AUC) and is plotted as column graphs. c , Oxidative burst of neutrophils initially cultured on glucose (10 mM; Glu and Glu + PMA groups) or other nutrients (10 mM) following stimulation with PMA. After calibration and three baseline measurements, PMA (100 ng/mL final concentration) was injected via the first injection port and OCR was measured. Approximately 100 min later, glucose (10 mM final concentration) was injected via the second port (final glucose concentration for Glu and Glu + PMA groups was 20 mM, all others were 10 mM) and OCR measured. The magnitude of the initial oxidative burst (UAC 1 st injection, PMA) was determined from the area between the first and second injections. The second oxidative burst (AUC 2nd injection, glucose) was determined from the AUC following the second injection. The overall oxidative burst (AUC Overall) is the sum of the two bursts. d , Neutrophil oxidative burst following Mtb infection (MOI of 5). Neutrophils cultured in medium containing 10 mM glucose were exposed to Mtb for 30 minutes, washed three times in glucose-free medium to remove extracellular bacilli, then cultured in medium containing 0-10 mM glucose. Uninfected controls were maintained in 10 mM glucose. AUC was calculated beginning with the first OCR measurement. e , Infection of neutrophils by injecting Mtb bacilli through XF injection port B in medium containing 10 mM glucose. After calibration and baseline measurements, rotenone (1.25 μM) and antimycin A (2.5 μM) were injected via port A to inhibit mitochondrial respiration. Mtb -GFP bacilli were injected through port B at an MOI of 1 or 5. After the completion of the XF run, Mtb -GFP distribution in the wells was visualized using the Agilent BioTek Cytation 5 Cell Imaging Multimode Reader to confirm uniform bacterial distribution. f , Oxidative burst of neutrophils cultured on glucose (0-10 mM) following injection of Mtb bacilli (MOI of 5) via port B. g, Oxidative burst of neutrophils cultured on glucose (10 mM) in response to live Mtb (MOI of 5), irradiated (irr.) Mtb , Mtb whole-cell lysate, or Mtb cellular fractions containing cell wall, cell membrane, or total lipids. h , Oxidative burst of neutrophils in medium containing various nutrients, with rotenone (1.25 μM) and antimycin A (2.5 μM) injected via port A prior to Mtb injection (MOI 1:5) via Port B. Glucose (10 mM final concentration) was later injected into all wells. i , Oxidative burst of neutrophils cultured in 10 mM glucose following infection with Mtb , M. avium , M. abscessus , S. aureus , or L. monocytogenes (all MOI of 5), via injection from port B. c–i, column graphs represent the magnitude of the oxidative burst and are shown as the mean ± SEM of the areas under the OCR curve for each group (n = 6-8 technical replicates per group). Statistical significance was determined using ordinary two-way ANOVA with Tukey’s multiple comparisons test (* P < 0.03, ** P < 0.002, *** P < 0.0002, **** P < 0.0001). All assays were performed independently three or more times.

Article Snippet: Liquid cultures of Mtb H37Rv and H37Rv-GFP, M. avium 2285 Rough (BEI Resources, NR-44264) and M. abscessus (Moore and Frerichs) Kusunoki and Ezaki (ATCC, cat # 19977) were grown at 37 °C with shaking in Middlebrook 7H9 broth (Thermo Fisher cat # DF0713-17-9) supplemented with 0.2% glycerol, ADS (albumin [Millipore Sigma cat # 3116956001], dextrose [Thermo Fisher cat # DF0155-17-4], and sodium chloride), and 0.02 % tyloxapol (Sigma-Aldrich cat # T8761).

Techniques: Infection, Software, XF Assay, Injection, Cell Culture, Concentration Assay, Imaging, Irradiation, Membrane